I. Field of the Invention
The present invention relates generally to the field of animal sciences. More specifically, the invention relates to methods and compositions for detecting early stage pregnancy, relating to proteolytic activity.
II. Related Art
Pregnancy diagnosis is a critical aspect of sound reproductive management in the cattle (e.g. dairy and beef) industry. In general, artificial insemination is successful less than 50% of the time and the producer must either rely on overt signs of return to estrus (that are easily missed) or delay rebreeding until pregnancy failure is confirmed by ultrasound, palpation or by assay of circulating pregnancy-specific antigens. Such delays are extremely costly and constitute a major economic loss to the industry.
An accurate pregnancy test for cattle which can be performed early in pregnancy has long been sought. Several pregnancy tests are available, including a milk progesterone assay (Oltenacu et al., 1990; Markusfeld et al., 1990), estrone sulfate analysis (Holdsworth et al., 1982; Warnick et al., 1995), rectal palpation (Hatzidakis et al., 1993), and ultrasound (Beal et al., 1992; Cameron and Malmo, 1993), or by assay of pregnancy-specific antigens, among others.
Pregnancy-associated glycoproteins (PAGs) are a family of proteins structurally related to aspartic proteinases such as pepsins, and are expressed in the outer epithelial cell layer (trophoblasts) of the placenta of even-toed ungulates (Green et al., 2000; Hughes et al., 2003; Xie et al., 1997a). Ruminant PAGs have not yet been shown to be catalytically active as proteinases, although each appears to possess a cleft capable of binding peptides (Guruprasad et al., 1996). It is estimated that cattle, sheep, and most probably all ruminant Artiodactyla possess dozens of PAG genes. Other artiodactyls, such as swine, also have PAG genes. Polypeptides with somewhat related sequences are also known to exist in species outside the Artiodactyla, including horses, cats and rodents.
Many members of the ruminant PAG family are able to enter the maternal circulation during pregnancy. Due to the placenta-specific origin of these proteins, their presence in maternal blood can serve as a means of determining pregnancy status of bred cattle. Indeed, immunological-based assays have been used as the basis for making such determinations (e.g. U.S. Pat. No. 6,869,770).
Phylogenetically, the ruminant PAGs are a highly diverse family that fall in two distinct groupings: the more recently evolved ‘modern’ PAGs and the ‘ancient’ PAGs. Even in initial protein purification studies (Butler et al., 1982; Zoli et al., 1991; Xie et al., 1991; Xie et al., 1994; Xie et al., 1996), it was clear that the boPAGs were heterogeneous in molecular weight and charge, and as more isoforms were purified, it was evident that they differed in their amino terminal sequences (Atkinson et al., 1993; Xie et al., 1997a). Despite their relationship to aspartic proteinases, many PAGs, particularly those in the modern grouping, are incapable of proteolytic activity due to site-specific mutations within the catalytic site (Green et al., 1998, Telugu et al., 2005).
Several peptidases have been shown to be products of the placenta or of the uterine decidua. Some, such as pro-renin, placental leucine aminopeptidase, pregnancy-associated plasma protein A (IGF binding protein 4-peptidase) and neutral IGFBP-3 protease, are able to enter the maternal circulation (Bischof, 1989; Poisner, 1998; Irwin et al., 2000; Bischof, 2001; Nomura et al., 2005). Often, the activity of these peptidases in serum is monitored by zymography. The proteolytic activity of neutral IGFBP-3 protease (as measured by the cleavage of a specific protein substrate (IGFBPs)) has been shown to increase in maternal serum throughout the course of pregnancy (Irwin et al., 2000). Other clinical measures of enzymes circulating in the blood (some being peptidases) have been used to monitor various physiological states (Murooka et al., 2001; Clements et al., 2004; Wu et al., 2004; Deegan et al., 2005; Sidikou et al., 2005; Zitouni et al., 2005; Villanueva et al., 2006). However, there are no known reports that describe the measurement of proteolytic activity as a means for pregnancy diagnosis in ungulates. Thus there remains a need to provide additional assay platforms for accurate and early detection of pregnancy, especially in agriculturally important animals such as cattle.